This invention is directed to chimeric recombinases incorporating a novel DNA binding domain preferably but not limited to at least one zinc finger domain and at least one domain that has catalytic activity that promotes recombination and methods for optimizing the activity of these recombinases by directed evolution, as well as to applications of the chimeric recombinases and the methods in gene therapy and the modification of DNA in other organisms, for example for endowing crop plants, animals and industrial organisms with favorable phenotypes.
At present, no strategy for gene therapy enables targeted and site-specific recombination of the endogenous human genome. Such a strategy would allow the rapid excision of harmful genes and the safe integration of beneficial ones.
The Cre-loxP recombination system enables researchers to efficiently alter the genome of discrete cells in vivo. Once genomic lox sites have been introduced by homologous recombination, the Cre recombinase may catalyze excision, inversion, or integration, at those loci. This revolutionary tool continues to find novel applications including circumvention of embryonic lethality with induced gene inactivation and delineation of cellular lineages during embryogenesis (16). With the development of Cre, the Flp recombinase and the φC31 integrase, site-specific recombinases (SSRs) now comprise a toolbox for genetic manipulation.
True to their name, SSRs are highly specific for the ˜28 bp recombination sites present in their native substrates. While a few mutant recombination sites have been found to be functional, this fundamental requirement broadly prohibits the application of SSRs to endogenous genomes. Constrained by the prerequisite of homologous recombination, SSRs are barred from many potential applications, gene therapy being perhaps the most significant. This constraint has motivated several groups to modify SSR substrate specificity by directed protein evolution (18, 53, 54). Calos and coworkers characterized “pseudo” attP sites within the endogenous human and Mouse genomes at which φC31 mediates efficient integration (65). Their application of this enzyme to the treatment of junctional epidermolysis bullosa (48), Duchenne muscular dystrophy (50), and murine hereditary tyrosinemia type I (31) suggests the therapeutic potential of endogenous site-specific recombination.
The extent to which Cre and φC31 can be trained on new substrates is limited by the structural organization of their DNA binding interactions. Tyrosine recombinases, such as Cre, mediate DNA binding and catalysis with the same protein domain. This arrangement constrains the geometry of all potential DNA-protein interactions and precludes replacement with an exogenous DNA binding domain. Notably, the characterization of one mutant Cre-substrate interaction revealed recognition to be indirect —with contact to the altered base pair mediated by a bridging water molecule (7). In contrast to the well characterized tyrosine recombinases, the function of the φC31 integrase, and other large serine recombinases, remains largely obscure. In the absence of a three dimensional protein structure or known DNA binding domains, Calos and coworkers evolved φC31 by covering the entire protein sequence with random mutations (54). Modification of the large serine recombinases is further complicated by the potential multiplicity of significant DNA binding regions (2).
Accordingly, there is a need for a more generalized method of catalyzing targeted and site-specific recombination of the endogenous genome, particularly for gene therapy, as well as for enzymes that can catalyze such targeted and site-specific recombination. This is particularly useful for gene therapy, but would have many other applications in molecular biology, including in gene cloning and use in modification of industrial organisms and agricultural plants and animals.